An overview on mRNA-based vaccines to prevent monkeypox infection

The human monkeypox virus (Mpox) is classified as a member of the Poxviridae family and belongs to the Orthopoxvirus genus. Mpox possesses double-stranded DNA, and there are two known genetic clades: those originating in West Africa and the Congo Basin, commonly known as Central African clades. Mpox may be treated with either the vaccinia vaccination or the therapeutics. Modifying the smallpox vaccine for treating and preventing Mpox has shown to be beneficial because of the strong link between smallpox and Mpox viruses and their categorization in the same family. Cross-protection against Mpox is effective with two Food and Drug Administration (FDA)-approved smallpox vaccines (ACAM2000 and JYNNEOSTM). However, ACAM2000 has the potential for significant adverse effects, such as cardiac issues, whereas JYNNEOS has a lower risk profile. Moreover, Mpox has managed to resurface, although with modified characteristics, due to the discontinuation and cessation of the smallpox vaccine for 40 years. The safety and efficacy of the two leading mRNA vaccines against SARS-CoV-2 and its many variants have been shown in clinical trials and subsequent data analysis. This first mRNA treatment model involves injecting patients with messenger RNA to produce target proteins and elicit an immunological response. High potency, the possibility of safe administration, low-cost manufacture, and quick development is just a few of the benefits of RNA-based vaccines that pave the way for a viable alternative to conventional vaccines. When protecting against Mpox infection, mRNA vaccines are pretty efficient and may one day replace the present whole-virus vaccines. Therefore, the purpose of this article is to provide a synopsis of the ongoing research, development, and testing of an mRNA vaccine against Mpox.


Introduction
The monkeypox virus (Mpox) may be an uncommon member of the Poxviridae family and the Orthopoxvirus genus.The West African and Congo Basin clades, often known as the Central African clades, are two genetically distinct Mpox clades.Mpox possesses double-stranded DNA (dsDNA) [1].At the State Serum Institute of Copenhagen, the virus was discovered in monkeys in 1958 and was named "Mpox" [2].In August 1970, a 9-year-old boy with a fever who was appealed to Basankusu Hospital in the Democratic Republic of the Congo (DRC) was found to have the first Mpox infection [3].In 2003, the United States (U.S.) Midwestern region had the first Mpox outside Africa.Instead of monkeys and squirrels as reservoirs in this outbreak, prairie dogs, native to the U.S., were named as reservoirs.Infected Gambian pouched rats transmitted the Mpox virus in the prairie dogs into the nation from Ghana [4].Additionally, there are still many unknowns regarding the Mpox's evolutionary history and the timing and location of viral diversification in Africa [5].The World Health Organization (WHO) states that as of July 20th, 2022, an Mpox outbreak had spread to more than 82 locations after first being recognized in May 2022 in the United Kingdom [6].This epidemiological research reports on 508 confirmed human cases of Mpox during the first 5 weeks of the Madrid outbreak in 2022.In this large outbreak, the occurrence of almost all cases (99%) in men who have sex with men (MSM), together with the predominant location of the rash in the genital, perineal, or perianal area and the presence of lymphadenopathy in the inguinal region, indicate that close physical contact during sexual activities has been highly involved in the transmission of the infection in this outbreak [7,8].A recent pandemic of Mpox was detected in all six WHO regions in May 2022.The WHO declared it a public health emergency on July 23, 2022.Before the current pandemic, Mpox had been documented in individuals residing in various regions of central and West African countries.Furthermore, the vast majority of Mpox cases in non-African populations were attributed to international travel to countries where the disease is prevalent or to the importation of livestock.As immunity induced by the smallpox vaccine declines, Mpox has the potential to infect the entire global population.While the virus does not typically induce significant mortality in individuals with healthy immune systems, severe illness and death may ensue if it were to transmit to immunocompromised individuals, children, the elderly, pregnant women, or those with comorbidities including diabetes.The dissemination was hypothesized to have transpired via intimate contact in 95% of the infected individuals.It was discovered that 41% of those infected with HIV were homosexual or bisexual males, comprising 98% of those infected [9].Moreover, by 2022, 3-6% of diagnosed cases had passed away.The initial documented case of Mpox outside of Africa occurred in the Midwestern United States in 2003.A total of seventy-one individuals contracted the infection; however, there were no reported fatalities.In 2005, a total of 49 cases were documented in Sudan; a genetic analysis study subsequently unveiled that the virus's source was likely not Sudan but rather the DRC.Significantly more measles cases have been documented in Africa, with the DRC reporting approximately 2,000 cases annually from 2011 to 2014.Nigeria recorded a total of ten human-Mpox infections between 1971 and 1978.In September 2017, 118 verified cases were documented; in September 2018, the initial case was reported in the United Kingdom.Cases of the current outbreak were reported outside of endemic regions in Europe, Oceania, Asia, and the Americas since May 2022.A total of 16,000 individuals have been verified dead in over 70 countries; the current death toll stands at 0.03% [10].The virus's geographic dissemination may be ascribed to the importation or international transportation of infected animals from countries afflicted with the disease.Mpox, which has manifested globally as cluster outbreaks, has not only captured the attention of the WHO but also prompted international health organizations to collaborate extensively and with heightened vigilance.Initial findings derived from the sequencing of the genomes of Mpox DNA isolates currently prevalent in different countries suggest that the outbreak affecting multiple countries may be attributable to the West African clade.It is noteworthy that Mpox genome sequences acquired from multiple nations have exhibited divergence from the clade found in West Africa.However, further research is required to determine the impact of these mutations or genomic alterations on the transmissibility, virulence, and immune evasion of viruses [11].While Mpox symptoms frequently endure for two to four weeks and are self-limited, severe cases can occur and have a case fatality rate of three to ten% [12].However, several licensed medications and vaccines are only given to people with serious illnesses or impaired immune systems.There is no effective and secure treatment for Mpox infection [13].Vaccination against smallpox has halted in most communities and countries, resulting in dwindling immunity.Despite evidence that the smallpox vaccination protects against Mpox by 85%, immunization against the smallpox virus has not been offered since the WHO declared the smallpox virus extinct in 1980.Furthermore, in pregnant women, transmission through the placenta has been linked to congenital Mpox, which can manifest during and after childbirth and in the newborn.The duration of the protection is yet unclear.The outbreak will be contained and prevented from spreading further within and outside the initial high-risk group if vaccination decreases sexual Mpox transmission [14,15].Treatment with antiviral medications of individuals already infected with a virulent orthopoxvirus would provide immediate benefit, as opposed to the vaccine, whose protective impact is delayed.The effectiveness of antiviral drugs in controlling an outbreak has not been examined because they were not accessible during the smallpox eradication campaign.Antiviral therapy would undoubtedly be advantageous without immunization for treating Mpox patients and for containing the spread of illness [16].Numerous nucleic acid test techniques have also been created to detect and characterize the Mpox.DNA polymerase (E9L) and envelope protein (B6R) are two of the orthopoxvirus genes that are the focus of the experiments [17].No specific treatments for diseases brought on by the Mpox have yet received Food and Drug Administration (FDA) approval.However, there are several antiviral drugs (Tecovirimat, Cidofovir, Brincidofovir, and Vaccinia Immune Globulin Intravenous (VIGIV)) that were created to treat smallpox that is now being used to treat Mpox.However, no information exists on their efficacy in treating infections caused by Mpox [18].
Given the WHO's proclamation of Mpox infection as a worldwide public health emergency, nations must prioritize the timely and comprehensive evaluation of efficacy and effectiveness to ensure preparedness for prompt vaccination implementation.The WHO issued interim recommendations on Mpox immunization on June 24, 2022, stressing that widespread vaccination is not necessary nor advisable at this time [19].The use of standardized procedures and data-gathering methods is of utmost importance in collaborative research about vaccine efficacy.WHO places significant emphasis on the provision of support for vaccination initiatives, which includes surveillance and contact tracing.Furthermore, the WHO emphasizes the need to implement efficient public health communication and robust pharmacovigilance within this particular environment [20].The JYNNEOS vaccine has been authorized for use in smallpox and Mpox prevention during the current disease outbreak in the US.JYNNEOS can be replaced with ACAM2000, which has been approved to aid smallpox and Mpox prevention [21].During phase III clinical trials, Modified Vaccinia Virus Ankara (MVA) showed a favorable safety profile.However, it is essential to note that further vaccine studies on a larger scale are still required before MVA can be licensed for widespread use in the general population as a preventive measure against smallpox and Mpox infections.The pricing of the product is one of the factors to consider.Therefore, postexposure vaccination against Mpox and smallpox with ACAM2000 or MVA remains the preferred option [22].Despite their potential, the ACAM2000 and MVA vaccines have not met the worldwide medical need.New research has shown that vaccination with JYNNEOS only partially increases the production of neutralizing antibodies (nABs) against Mpox.Therefore, to combat the current Mpox, a safe, effective, and readily accessible Mpox-particular vaccine is urgently needed [23].A safe and effective vaccine against the disease is possible using nucleic acid vaccines.Vaccine production on a massive scale is also easy and cheap.mRNA vaccines provide many advantages in preventing viral infections.These include their ability to be synthesized quickly and on a large scale, their outstanding safety record without the need for nuclear entry, and their efficacy in stimulating both humoral and cellular immune responses [24,25].Once inside, cells interpret the mRNA as a set of instructions and construct proteins that bind to antigens on the pathogen.The immune system recognizes these foreign antigens as invaders, mobilizing defenses known as ABs and Tcells and preparing the immune system for possible future assaults [26].Stanford Medicine investigators have discovered that the Pfizer/BioNTech mRNA vaccine, designed to combat COVID-19, exhibits superior efficacy in stimulating killer T cells, a crucial component of the immune system, compared to natural infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19) [27][28][29].Multiple studies have provided evidence supporting the advantageous use of lipid nanoparticles (LNPs) as a delivery mechanism for mRNA vaccines, owing to the notable advancements achieved in suppressing Mpox.The reason for this phenomenon is that nanoparticles (NPs) are well recognized as a reliable mechanism for continuous delivery, ensuring the preservation of mRNA integrity, enhancing cellular absorption, and improving the efficiency of transfer and release of nucleic acids inside host cells [30].Researchers should also advance knowledge of Mpox, its clinical management, and infection control and prevention expertise, particularly among public health personnel.Equal access to medical care and vaccines should be guaranteed, and discrimination and stigma within the MSM community should be adequately addressed.Finally, researchers should start an international partnership to perform clinical studies to examine the effectiveness and safety of Mpox vaccines and antiviral medications [31].
Considering the existing conditions regarding the Mpox, Specific focus should be given to vaccinating this virus.Accordingly, in this study, we will examine the different characteristics, prevention methods, and different designed mRNA vaccines for Mpox.

Characteristics and structure of Mpox
The first cases of human-Mpox infection were reported in Africa in 1970.The orthopoxvirus's natural reservoir was small rodents, with humans and primates as accidental hosts.Cross-protection against Mpox is induced by smallpox immunization.Six decades ago, the Global Smallpox Eradication Program (GSEP) and Mpox coexisted in Africa.An assessment of the literature about the possible effects of Mpox on GSEP initiatives was spurred by the human Mpox outbreak of 2022.Because the two orthopoxviruses are identical, proving that there isn't a non-human smallpox reservoir was crucial to evaluating the GSEP.A non-human smallpox reservoir was discovered to be very far away.Human Mpox was restricted to non-vaccinated individuals and did not manifest in smallpox-vaccinated humans.Before 1989, surveillance in the DRC determined that Mpox was highly improbable to persist in humans and to develop into a significant public health concern.Decades after smallpox vaccination ceased, Mpox surveillance in the DRC from 2005 to 2007 revealed a 20-fold increase in the incidence of human Mpox, which was correlated with much-reduced rates of prior smallpox vaccination [32].Smallpox was exclusively transmitted between humans and lacked a recognized zoonotic host; conversely, Mpox could be transmitted to humans via animal hosts.Human Mpox was intermittently present in rural communities across West and Central Africa, where serological surveys were sponsored by the WHO.Preceding 1986, population-based surveys estimated that 12-15% of children exhibited ABs to the orthopox virus.Patients had a mean age of merely four and a half years.245 of 338 cases of Mpox were attributed to animal sources [33].The trade-in of rodents imported from Ghana to sell them as exotic pets sparked the outbreak.There is speculation that these rodents transmitted the infection to co-housed prairie dogs, also purchased as pets via animal-to-animal contact.Although there were 47 confirmed and probable cases reported, no fatalities occurred.The majority of the lesions were isolated cutaneous lesions.Vaccination against smallpox (DryVax, Wyeth) was provided to individuals who had direct contact with human cases and commercial rodents.Humanto-human transmission may have been a factor in two cases of the 2003 outbreak in the United States; however, contact with infected rodents remains possible [34].
Under an electron microscope, Mpox has a recognizable oval or brick-shaped structure with a lipoprotein envelope that ranges in size from 200 to 400 nm.Despite being a DNA virus, Mpox completes its life inside infected cells after binding on glycosaminoglycans and entering the host cells.To enter the host cells, it has also been proposed to use the classical apoptotic mimicking mechanism [35,36].Other methods include endosomal uptake using an actin-based macropinocytosis mechanism or by the interaction of ligands on the viral envelope and the plasma membrane receptors of the host cell, such as heparan sulfate or chondroitin sulfate, which causes fragments of the viral envelope to spread throughout the plasma membrane.The virus then secretes enzymes and viral proteins into the cell cytoplasm, which weaken cellular defenses and promote early gene expression, resulting in the development of early proteins, DNA replication, and the creation of intermediary transcription factors [37].The outer membrane, which has a lateral body on each side, protects the membrane bonds, an enzyme-filled core densely packed with a dsDNA genome and transcription factors [35,36].
Previous studies have shown that the Mpox genome has a high degree of similarity, about 96.3%, with the smallpox genome.This similarity is attributed to essential enzymes and proteins within the Mpox genome [38].Additionally, over 60 amino acid residues in 190 open reading frames (ORFs) were discovered [5,36].At nucleotide locations 56,000-120,000, Mpox, a muchconserved central coding region sequence (CRS), is surrounded by various ends, including inverted terminal repetitions (ITRs).The Mpox genome's ITR region has at least four identified ORFs [36].Mpox virions contain more than 30 membrane and structural viral proteins and transcriptional enzymes related to DNA-dependent RNA polymerase [39].The virus has two distinct infectious forms, namely extracellular enveloped virus (EEV) and intracellular mature virus (IMV).EEV is believed to play a crucial role in early dispersion, whereas IMV is produced upon cell lysis.IMVs and EEVs are fundamentally very different because IMVs lack the extra outermost membrane layer and infect cells in different ways (Fig. 1).However, the two forms of virions have various viral protein incorporation levels.Although Mpox replication is complicated, it is widely accepted that it is the same as other orthopoxviruses [39,40].

Immune responses in Mpox infection
The identification and eradication of Orthopoxviruses are facilitated by innate and adaptive immunity.Numerous DNA-sensing systems, such as DNA-dependent protein kinase, Toll-like receptor 9 (TLR-9), cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, cGAMP) synthase, and Interferon gamma (IFN-γ) inducible protein 16, are used by orthopoxviruses to trigger an immunological response.The nuclear factor kappa B (NF-κB) and IFN signaling pathways are activated due to the DNA sensors activating the Stimulator of Interferon Genes (STING).Orthopoxviruses generate intermediates of dsRNA that stimulate TLR3 and protein kinase R (PKR).The NF-κB and IFN pathways are activated by PKR and TLR.The 2 A complex of eukaryotic translation initiation is also phosphorylated by PKR, suppressing mRNA translation by this mechanism [42].After the identification of a virus and the initiation of an early innate response, the subsequent eradication of the virus heavily relies on the adaptive immune response.The majority of hematopoietic antigen-presenting cells (APCs) are CD14 + monocytes.The cells can display Mpox antigens to both CD4 + and CD8 + T cells, therefore facilitating the orchestration of cytokine production, the elimination of infected cells, and the serologic response of the host [42].Monkeys pulmonary and mediastinal lymphatics, as well as sinuses of infected lymph nodes, were teeming with immunopositive monocytic cells for poxviral antigens, indicating that these cells were the primary vehicle for lymphatogenous and subsequent hematogenous propagation.Lesions of the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver appeared to result from systemic viral dissemination via monocytic cellassociated viremia.It is postulated by researchers that the mononuclear phagocyte system plays a crucial role in facilitating the spread of the virus to secondary and tertiary locations throughout the body during systemic infection.Mpox antigen was only seen in tissues exhibiting morphological defects, mainly localized within fibroblasts, macrophages, dendritic cells (DCs), and epithelial cells of compromised tissues [43].Natural killer cells (NK cells), like monocytes, play a critical role in innate immunity and may influence the adaptive immune response.NK cell role in controlling Mpox viral load was revealed in CAST/EiJ mice, however, there was no correlation between viral clearance and NK cell numbers or activity.This strain is especially vulnerable to orthopoxvirus infection because of a lack of NK cells.A recent study has shown that the number of NK cells rapidly increases in the lymph nodes and peripheral blood of rhesus macaques infected with Mpox (by a mean of 46.1-fold by days 8-9 post-infection and 23-fold by day 7 post-infection, respectively).The infection caused by Mpox significantly impaired the migratory capacity of many subsets Fig. 1 A cell infected by the dsDNA Mpox will have both an intracellular mature virus (IMV) and an intracellular enveloped virus (IEV).Mpox is among the most sizable and intricate viruses, characterized by a brick-shaped architecture spanning 220-450 nanometers in length and 140-260 nanometers in width.Core, lateral structures, outer membrane, and outer lipoprotein envelope are its four constituent parts.The core, composed of double-stranded viral DNA and core fibrils, is the central component.The palisade layer is an impermeable structure that encircles this particular layer.The central nucleus, lateral bodies, and palisade layer are all contained within the outer membrane [41] of NK cells before their rapid proliferation, resulting in a detrimental effect on their recruitment to lymphoid and/ or inflammatory regions.Furthermore, it was shown that chemokine receptors such as CXCR3, CCR5, CCR6, and CCR7 exhibited a downregulation.Moreover, it has been demonstrated that NK cells, which are separated from blood and lymph nodes, have a reduction in their ability to undergo degranulation and produce IFN and Tumor necrosis factor (TNF).IL-15 treatment demonstrated efficacy in preventing deadly Mpox infection in CAST/ EiJ mice76, even in cases where CD4 + and CD8 + T cell numbers were reduced.This implies that the observed protective effect may be attributed to the expansion of NK cells.The administration of IL-15 treatment has been seen to induce a transient elevation in the population of CD8 + T cells and NK cells that exhibit the secretion of IFN [40].The two clades of Mpox are different in terms of pathogenicity.The Central African clade causes more severe diseases with excellent case fatality rates.The significant pathogenicity of this pathogen is attributed to its ability to hinder T cell receptor-mediated T cell activation and impede the production of pro-inflammatory cytokines such TNF-α and IFN-γ by human cells.The Central African clade also has a gene that prevents complement enzymes from working, which results in a critical immune-modulating component that contributes to its higher virulence.However, research has demonstrated that major histocompatibility complex (MHC) expression or cellular transit of MHC molecules is not affected by the virulence of Mpox [37].The West African clade's lower virulence is caused by deletions and fragmentations in the ORF [44,45].
As a member of the Orthopoxvirus genus, the smallpox virus induces a severe and highly contagious illness in humans.In 1980, smallpox was deemed extinct due to a worldwide vaccination campaign that utilized vaccines derived from the closely related vaccinia virus (VACV).There are no known specific interventions for Mpox infection in humans.In contrast, empirical evidence from Zaire during the 1980s indicated that individuals immunized against smallpox during the eradication effort also exhibited significant cross-protection against Mpox infection.Nevertheless, a substantial proportion of individuals aged 50 and below currently lack a record of smallpox vaccination, and the vaccines that were initially employed during the worldwide eradication effort are presently unavailable.Amidst the worldwide Mpox outbreak of 2022, more than one million doses of modernized smallpox vaccines were made available for preand post-exposure prophylaxis to populations classified as having a heightened susceptibility to Mpox [46].As immunity to the smallpox vaccine diminishes, Mpox has the potential to increase throughout the world's population [9].In China 42 years ago, the vaccinia virus Tiantan type (VTT) was used to protect people from getting smallpox.It is crucial to find out how immune people are to smallpox who were vaccinated 43 years ago or more and how susceptible their immune systems are to Mpox.A study used 294 volunteers to find out the level of remaining humoral immunity.They looked at the vaccinia-specific IgG level, the nAB titer, and the cross-ABs of Mpox A29L, B6R, A35R, and M1R.Results demonstrated that the population retains humoral immunity to the smallpox vaccine, whereas VTT-specific NAb levels decrease with age.Before 1981, a significant proportion of the populace that ought to have received VTT remained immunized against Mpox, specifically ABs that target A35R and B6R antigens.Based on these results, it appears that most Chinese populations retain VTTspecific IgG ABs for a minimum of 42 years following smallpox vaccination.These ABs may confer a degree of protection against Mpox [47].Researchers discovered that 60 (89.6%) and 40 (70.1%)vaccinated people had anti-Mpox IgG and Nabs, respectively.In 30% of vaccine recipients, researchers saw a T cell response to orthopoxviruses and Mpox peptide pools.Thus, research demonstrates that a significant percentage of persons vaccinated against smallpox 40-60 years ago exhibit humoral crossimmunity.However, a smaller subset (30%) of vaccinated individuals showed a T-cell-specific response to Mpox [48].
During the COVID-19 pandemic, novel variants of the SARS-CoV-2 virus emerged that exhibited the capacity to evade a portion of the immunity acquired from prior infection or the initial vaccines.This prompted the development of updated vaccines that could provide cross-protection against Omicron and other variants of the SARS-CoV2 virus that are cause for concern.It is unknown whether comparable mutations are occurring in the Mpox genome and whether future vaccine modifications will be necessary to maintain immunity.Nine people were found to have had breakthrough Mpox six months after immunization, according to French research conducted in the Loire Valley.It is unclear if this is a result of declining immunity, the comparatively low levels of Mpox-specific nAB responses brought on by the MVA-BN vaccine, or whether it portends the appearance of novel Mpox variants capable of eluding vaccineinduced immunity.Pre-exposure prophylactic studies have shown a limited number of breakthrough cases, which are to be anticipated despite vaccination's estimated ~ 85% effectiveness.Sagy et al., for instance, found five cases of breakthrough Mpox between 21 and 47 days after a single dose of vaccination [27,[49][50][51].
In addition, the Mpox virus has been identified as possessing the ability to encode a diverse range of viral proteins that play a crucial role in circumventing the host's immune response [52].These substances can disturb critical transcription factors involved in synthesizing genes associated with inflammation, such as interferon regulatory factor 3 (IRF3) and NF-κB, and impede the signaling pathway of receptors responsible for pathogen identification.As well as lowering IFNα/β synthesis and obstructing protein kinase R (PKR)-mediated pathways, Mpox can also interfere with interferon signaling.Mpox also secretes proteins targeting important inflammatory molecules like TNF, IFN-γ, IL-1β, IL-18, and IL-6 [40,53].Moreover, Mpox can inhibit apoptosis in infected cells by synthesizing several viral proteins that disrupt the apoptotic pathways [40].Another example of a Mpox immunomodulator is the complement control protein (CCP), which stops the complement activation pathway from starting [54].D14, which inhibits complement activation, is also expressed by the Mpox Zaire strain from Central Africa.However, this viral protein is not typified by the West African Mpox strain.Finally, Mpox may inhibit immune cell function by preventing T cell and NK cell activation [40,54] (Fig. 2).

Mpox infection vaccines
By protecting against severe illness and lowering hospitalizations, vaccination is seen as a crucial way to combat Mpox [57].Nevertheless, immune responses to one orthopoxvirus can detect additional orthopoxviruses.The level of protection resulting from this discovery might vary depending on the degree of relatedness  12).A greater total AB titer is seen, along with increased levels of IgG and IgM [55,56] between different orthopoxviruses.A population growing more immunologically naive is thought to be the cause of the rise in Mpox incidence since the end of smallpox immunization [45,[58][59][60].The ability to develop multiple animal models of smallpox infection for the testing of vaccines and antivirals was made possible by immunological cross-reactivity.Two causes are most responsible for this cross-reactivity.First, there are a lot of common immune epitopes because orthopoxviruses share many sequence similarities, especially when it comes to immunologically significant proteins.Furthermore, at least 24 membrane and structural proteins are targeted by the ABs [60].There is evidence to suggest that the smallpox vaccine may provide some protection against Mpox and alleviate its clinical manifestations.There are three smallpox vaccines in the US Strategic National Stockpile (SNS) at present: ACAM2000®, JYNNEOSTM (also known as IMVAMUNE, IMVANEX, MVA-BN), and the Aventis Pasteur Smallpox Vaccine (APSV), which may be used for smallpox under an investigational new drug (IND) procedure [61].
In 1980, the WHO declared the global eradication of naturally occurring smallpox.However, subsequent apprehensions over bioterrorism and outbreaks of Mpox prompted the authorization of further smallpox vaccinations based on VACV in the early years of the twenty-first century.The first smallpox vaccines, such as Dryvax, were developed using the lymph-derived Lister/Elstree, Ikeda, and New York City Board of Health VACV strains, which were cultured on animal skin.In contrast, the development of the 2nd and 3rd generation vaccines included using cell culture technologies, which were employed to enhance their safety profile.Currently, inside the US, there are two officially authorized smallpox vaccines.The US FDA approved ACAM2000, a second-generation smallpox vaccine based on a replicating VACV, in 2007.ACAM2000 is based on a Dryvax clone.Those identified as having a heightened susceptibility to the smallpox virus should undergo active vaccination as a preventive measure.However, it is not suggested to pursue immunization specifically targeting Mpox in this population.The administration of ACAM2000 is contraindicated in individuals with high immunosuppression because of the potential for notable adverse reactions, such as myopericarditis, particularly in those who lack prior exposure to smallpox.The creation of alternative vaccines based on further attenuated VACV has been achieved by using many passages of the virus in primary cell cultures or eggs as the attenuation technique [20].The year 2019 saw the approval of Jynneos by the FDA.Jynneos is classified as a live, non-replicating, attenuated immunization of the third generation, specifically designed for smallpox prevention.Following the confirmation of the first case of the ongoing Mpox epidemic in the U.S. on May 17, 2022, the administration of the JYNNEOS vaccine (an MVA vaccine developed by Bavarian Nordic) was initiated as a preventive measure against the illness.On June 28, 2022, the U.S. National Immunization Plan proposed the administration of subcutaneous vaccines to individuals who have been exposed to or are suspected of being exposed to Mpox.This recommendation aims to broaden the scope of individuals eligible for immunization as part of an expanded postexposure prophylaxis (PEP) strategy.The FDA issued an emergency use authorization (EUA) on August 9, 2022, allowing the intradermal delivery of 0.1 mL of JYNNEOS, augmenting the available vaccine inventory [62][63][64].Patients who received JYNNEOS were not at risk for experiencing severe side effects such as myopericarditis or cardiomyopathy.ACAM2000 vaccines, however, can have severe side effects, such as breathing problems, facial swelling, lightheadedness, and an elevated risk of myopericarditis and cardiomyopathy.ACAM2000 and JYNNEOS are vaccines that the US FDA has approved.However, ACAM2000 has a higher risk of significant side effects, including cardiac issues, while JYNNEOS has fewer.As a result, JYNNEOS is preferable to ACAM2000.Further, JYNNEOS is advised for preexposure prophylaxis against orthopoxvirus infection in individuals at risk of exposure to Mpox illness [64,65].The APSV, a replication-competent vaccinia vaccine, may be administered under an Investigational New Drug Application (IND) or EUA when the licensed immunizations are unavailable or unsuitable.It is expected that the safety profiles of ACAM2000 and APSV would be comparable.It was also hypothesized that APSV, like ACAM2000, would result in myopericarditis.The US FDA has given conditional approval to the use of APSV, an IND, or an EUA in situations where ACAM2000 is either not appropriate or unavailable [61,66,67].
The use of the Lister (Elstree) strain of vaccinia has been instrumental in developing the LC16 m8 vaccine, a live, replicating, third-generation vaccination against smallpox.This particular vaccine has had official authorization for active immunization in Japan since 1975.In August 2022, the Japanese authorities broadened the vaccine's scope to include safeguarding against Mpox.These vaccines can be given to immunocompromised patients due to their better safety profile and attenuated phenotype.The effectiveness of these Mpox vaccines is determined by using data obtained from animal studies, which indicate the protection provided by immunization with these vaccines in non-human primates, as well as data from clinical trials that establish their immunogenicity in humans [20,68,69] (Table 1).

Novel vaccines for Mpox infection
The pursuit of developing novel vaccines to combat emerging and transmissible diseases is impeded by significant conceptual and scientific challenges.The COVID-19 pandemic has highlighted the challenges associated with developing vaccine formulations that are both safe and highly effective.These formulations are typically based on popular biological platforms, such as complete attenuated or inactivated SARS-CoV-2 virus, non-replicative human or simian adenovirus DNA vectors that utilize sections of the virus, or specific mRNA templates targeting portions of the viral spike (S) glycoprotein.To mitigate the risk of vaccine tampering and prioritize the safety of researchers, the current protocols for producing and distributing viral vaccines based on biological platforms need the use of highly regulated biosafety facilities, as well as stringent cold chains and packaging lines.Furthermore, due to their poor stability and specificity, second-generation vaccinations are not a practical approach toward subunit vaccines.On the other hand, controlled synthesis techniques have made it possible to generate vaccine formulations for numerous deadly illnesses, like malaria and COVID-19, that are low-cost, stable, specific, nontoxic, and safe [27,[76][77][78][79][80].Hence, exploring novel vaccine strategies targeting Mpox is essential to mitigate the risk of infection.Promising approaches include inactivated vaccines, live-attenuated vaccines, virus-like particles (VLPs), recombinant protein vaccines, nucleic acid vaccines, and NP-based vaccines.
Intracellular mRNA delivery is aided by the most advanced technology, which is led by LNPs.The most crucial component for mRNA production is the ionizable lipid, which is usually present in LNPs along with phospholipids, sterols, and lipid-anchored polyethylene glycol (PEG).Protein expression is not the only element that determines vaccine efficacy, according to that analysis.Other variables also play a role.NP biophysical characteristics are known to influence immunogenicity, as is the case with other vaccination methods.The size of NPs affects their ability to stimulate the immune system and their dispersion in tissues and cells when they are used as nanocarriers for protein-based vaccines [81,82].The size of the liposome is another factor that influences the T-helper to T-helper 2 ratio (Th1/Th2).Therefore, compared to vesicles of bigger or smaller size, those between 250 and 750 nm elicit stronger Th1 responses.On the other side, multilamellar vesicles could trigger a greater number of Th2 responses.It has also been shown that LNP's capacity to trigger the immune system is affected by their surface charge.For example, vesicles with a positive or negative charge elicit stronger antibody-neutralizing reactions compared to vesicles with no charge.Because of their interaction with innate immune system components, cationic lipids enhance the immunogenicity of LNP-formulated mRNA vaccines, resulting in adequate therapeutic effectiveness [83][84][85].The advantages of LNP over other mRNA delivery methods, such as the mononuclear phagocyte system (MPS), cellular uptake facilitation, minimal immunogenicity, and endosomal trapping, make it an up-and-coming candidate.Immune contact, including innate and adaptive immunity, and interaction with LNP components, was one of several obstacles encountered by mRNA-loaded LNP.The Vaccination Institution Ankara maintains the dermal vaccinia strain Ankara (chorioallantois VACV Ankara, or CVA), isolated from a horsepox viruslesion in Ankara, Turkey.This strain is the source of modified vaccinia Ankara.To prevent orthopoxvirus infection before exposure to those at risk of contracting Mpox disease, JYNNEOS is advised.[66,74] LC16m8 Third-generation Japan extended the indication of this vaccine (August 2022) To lessen the chances of problems caused by autoinoculation, a clone of LC16 was chosen for further study because of its ability to create microscopic pocks on chorioallantois membranes (CAM).Although it is associated with slower development in mammalian tissues, this trait is prized for prolonging the pock response.After six further passes in the primary rabbit kidney at a lower temperature, the virus was ready to be cultured on chicken embryo fibroblast cells.Clone 8 (or LC16m8) is the final attenuated strain.
Using information from animal studies, which shows protection against Mpox in non-human primates injected with these immunizations, researchers may make educated guesses about the efficacy of vaccines against Mpox.Additionally, clinical trial data is utilized to evaluate the vaccines' immunogenicity in human patients.
[66, 67,75] Two vaccines are available for reducing the risk and severity of Mpox infection in the United States: JYNNEOS® (Imvamune or Imvanex) and ACAM2000.JYNNEOS® is the preferred vaccine for the current outbreak of Mpox However, the LNP compositions highlighted a significant obstacle to the clinical delivery of mRNA to target tissues via cytosolic transport.As a result, they may reduce the likelihood of unwanted side effects by increasing vaccine absorption by DCs and blocking mRNA interactions with non-APCs [86].Understanding the mechanisms by which mRNA-LNP treatment induces these adverse effects and developing preventative measures is vital.Anti-PEG ABs are produced by the body in response to PEGylated LNPs, which may result in adverse effects.Important cells of the immediate hypersensitivity reaction, basophilic granulocytes or mast cells, are the sites where IgE ABs bind to FcεRI.As mediators, several tyrosine kinases are stimulated.The body binds anti-PEG IgM to the PEGylated liposome upon administration.This complex activates the classical complement pathway and rapidly exits the bloodstream due to Kupffer cell phagocytosis, a process known as the accelerated blood clearance (ABC) phenomenon.This anaphylatoxin stimulates basophils, mast cells, and macrophages, producing inflammatory mediators.This mediator activates complement (C) activation-related pseudoallergy (CARPA) via binding to the receptors of smooth muscle, endothelium, and autonomic effector cells.The following are some ways that mRNA-LNP-based medications might trigger autoimmunity: (1) The innate immune system responds to LNPs as an adjuvant, which causes the autoimmune process to proceed; (2) mRNA functions as an autoantigen and initiates the autoimmune process through TLR7; and (3) in the case of mRNA-LNP vaccines, the immune system is strengthened, potentially exacerbating the autoimmune response [87].[90].
In another study, J. W. Hooper et al. showed that upon an otherwise fatal assault with Mpox, rhesus macaques inoculated with a DNA vaccine composed of four VACV genes (L1R, A27L, A33R, and B5R) were shielded against severe disease.Animals immunized with a single gene (L1R), which specifies a pathogen-nAB target, incurred severe illness but lived.This is the first instance of the viability of a subunit vaccine strategy for smallpox-Mpox immunization [91].The Mpox E8L protein was shown to include an annular ganglioside-binding motif, according to a separate investigation.This motif is shared by three potential B linear epitopes that might be used to provide a safe and effective vaccine against Mpox.Since these three sequences were identified in the E8L protein, it was recommended that they be used as immunogens in a future Mpox-specific vaccine formulation (recombinant protein, synthetic peptides, or genetically based).This lipid raft/ganglioside-based strategy may offer therapeutic and vaccine responses to future virus epidemics in addition to present therapies [92].Based on the available data, there remains a requirement to advance the development of effective and secure novel vaccines designed explicitly for Mpox.This necessitates exploring innovative vaccine approaches, including VLPs, recombinant protein, nucleic acid (mRNA or DNA), and NP-based vaccines, before declaring Mpox as a [23,65,93].To enhance the specificity and efficacy of vaccines, researchers have developed subunit vaccines that focus on conserved antigens.These vaccines are administered in the form of purified proteins or plasmid DNA or through virally vectored vaccines.These approaches specifically target one or both of the two distinct immunological forms of poxviruses that cause infection, namely the mature virion and the enveloped virion [94].Nucleic acid vaccines have shown safety and efficacy comparable to those of inactivated vaccines, effectively emulating the vaccination process.Moreover, producing these vaccines using industrial means is both cost-effective and straightforward [95,96].To identify the most effective vaccine, it is crucial to evaluate many factors, such as the impact on the body, reactogenicity, safety profile, cytotoxicity, and potential side effects associated with vaccination, particularly among those at a higher risk or more susceptible to adverse outcomes.The outbreak of the 2022 Mpox in regions outside of Africa has brought attention to the lack of vaccinations that have shown efficacy and minimal adverse reactions.The use of this vaccine during the Mpox epidemic is believed to potentially contribute to the prevention or reduction of infection as pre-exposure prophylaxis (PrEP) for those near confirmed cases [97,98].

mRNA vaccines
The genetic instructions stored in the DNA in the nucleus are transported to the cytoplasm, where the ribosomes are responsible for translating them into proteins.mRNA treatments may restore protein activity for treating illnesses caused by the loss of particular protein functions, whereas most traditional medications operate by binding and blocking overactive disease-causing proteins.In addition, mRNA treatment is expected to have just the intended impact, as described by the nucleic acid sequence.mRNA is also simpler to generate and purify on a big scale than AB or cell treatments.In addition, mRNA is short-lived and cannot access the nucleus of a cell, making it very unlikely to result in genetic changes [99,100].The use of messenger RNA in vaccine development is also novel.Although mRNA vaccines are relatively new to the public, they have been under study by experts for quite some time.The DNA sequence encoding the spike protein serves as a template for the synthesis of the mRNA vaccine, which is then packaged into a lipoprotein-based carrier to facilitate its rapid uptake by cells and protection against destruction once within the body.When administered intramuscularly, the vaccine reaches more distant areas, and the mRNA molecules enter the cells, where they may help speed up the translation process.A humoral immune response will be triggered upon mRNA's entry into the body, prompting the maturation of B cells into memory B cells.In this way, memory B cells may effectively block antigens upon subsequent exposure.The two most common mRNA vaccines have been demonstrated to be safe and effective against SARS-CoV-2 and its many variants in clinical studies [101].Two mRNA-based vaccines, made by Pfizer-BioNTech (New York, NY, USA) and Moderna (Cambridge, MA, USA), have been authorized by the FDA.mRNA-1273, or "Spikevax, " is a rabies vaccine.EUA for mRNA-1273 in adults over the age of 18 was granted by the FDA [102].Conventional, nonreplicating, and self-replicating (self-amplifying) mRNA-based vaccines are the three main types.The mRNA used in nonreplicating constructs is short and basic, and it does not encode any proteins that may trigger an immune response inadvertently.The immunogen of interest is encoded inside a coding sequence flanked by 5′ and 3′ untranslated regions (UTRs), a 5' cap structure of 7-methylguanosine (m7G) linked to the first nucleotide through a triphosphate bridge, and a 3′-poly(A) tail.The 5′ m7G cap inhibits 5′-3′ exonuclease-mediated degradation, activates translation initiation factors, and promotes efficient translation while also preventing identification by the cytoplasmic RNA sensor, RNA helicases retinoic acid-inducible gene I (RIG-I).Maximal gene expression is also influenced by the size and organization of the 5' and 3' UTRs, as well as by regulatory elements in these regions.Both translation and stability of the mRNA vaccine construct depend on the poly(A) tail and its length.Sequence engineering (codon optimization) and nucleoside alteration (e.g., replacing uridine with pseudouridine) improve translation efficiency by reducing TLR recognition and the innate immunological response to mRNA constructions.Since tiny oligoribonucleotides and double-stranded RNA impurities are produced by DNA-dependent RNA polymerases during construct synthesis, mRNA purity is crucial.Protein translation and synthesis are stimulated by inhibiting the innate immune response and the generation of type I interferon and inflammatory cytokines, all of which are triggered when these pattern-recognition receptor-recognized contaminants are removed [103].However, mRNA in its natural state cannot be used therapeutically.The lack of an effective, well-tolerated delivery mechanism has been a major barrier to developing mRNA vaccines until recently.The need for cellular absorption and translocation is the main roadblock.The mRNA molecules face a severe barrier due to the negative potential across the cell membrane.Naked mRNA is too big and strongly negatively charged to passively penetrate the cell membrane, making it vulnerable to digestion by nucleases [104].
Self-amplifying mRNA and non-replicating mRNA vaccine constructions contain identical properties, including a 5′ cap sequence, 5′ and 3′ translated regions (UTRs), an open reading frame (ORF) carrying coding sequence (CDS), and a 3′ poly(A) tail [105].The versatility of plasmid DNA vaccines, together with improved immunogenicity and safety, is offered by self-amplifying mRNA vaccines.Reaching the cytoplasm of a cell, where the antigenic protein is encoded and amplified, is crucial for these vaccines to work as intended.Cellular absorption is hindered by RNA's hydrophilicity and large net negative charge.Physical delivery using electroporation or ballistic particles, as well as electrostatic complexation with cationic lipids or polymers, have been investigated as potential solutions to this problem to improve cellular absorption.Small animals and nonhuman primates have shown strong innate and adaptive immune responses in initial preclinical testing of self-amplifying mRNA vaccines delivered non-virally.Concerns about mRNA instability and the practicality of large-scale production have long cast doubt on the possibility of creating mRNA vaccines.In modern times, these concerns are seen as insurmountable obstacles to the technology's broad adoption.It is possible to manufacture nonamplifying mRNA vaccines in enough quantity and quality to satisfy regulatory standards, and they are now being studied in human clinical trials.If the promising results from human trials of self-amplifying mRNA vaccines are borne out by similarly excellent results in terms of immunogenicity, potency, and acceptability, this platform has the potential to establish nucleic acid vaccines as a flexible new tool for human vaccines [106].The future of non-virally administered self-amplifying mRNA vaccines is bright: they may be cheap, effective, easy to scale, and adaptable.The self-amplifying mRNA acts like a virus by amplifying its genome and the host cell's antigen-encoding mRNA.This causes the host immune system to mount a powerful and long-lasting response against the antigen, which includes both humoral and cellular immune responses.Furthermore, in theory, self-amplifying mRNA could encode any eukaryotic sequence without modifying the production process.This would allow for a more rapid and adaptable research and development timeline compared to current vaccines, allowing quicker response to new infectious diseases [107].The coding sequence (CDS) is encoded by non-replicating mRNA (NRM) constructs, which are surrounded by untranslated regions (UTRs) at the 5′ and 3′ ends, a 5′-cap structure, and a 3′-poly-(A) tail.The self-amplifying mRNA (SAM) construct contains supplementary replicase components that can regulate the amplification of mRNA within cells.NRM and SAM are formulated within LNPs, which serve to encapsulate the mRNA constructs, thereby facilitating cellular uptake and safeguarding against degradation.mRNA into cells via its delivery system is commonly transported via membrane-derived endocytic pathways.Endosomal escape facilitates the mRNA's entry into the cytosol.Localized within cytosol, ribosomes promptly transform NRM constructs into the target protein, which is then subjected to post-translational modifications.Ribosomes are also capable of promptly translating SAM constructs to generate the replicase apparatus essential for the selfamplification of the mRNA.Ribosomes facilitate the translation of self-amplified mRNA constructs into the target protein, which subsequently experiences posttranslational modification.The proteins under consideration are produced in intracellular, transmembrane, or secreted forms.Adaptive and innate immune responses identify the target protein [108].
mRNA treatments provide many advantages in terms of reduced risk of pre-existing or anti-vector immunity, enhanced safety, precise dosage control, and the potential for multiple administrations.Pardi et al. conducted an experiment where they administered a single dose of LNP-encapsulated nucleoside-modified mRNAs encoding the heavy and light chains of the anti-HIV-1 nAB VRC01 via intravenous injection in mice.The researchers observed significantly elevated levels of functional AB in the serum following this administration.Moreover, this intervention effectively protected the humanized mice from HIV-1 infection [24,109,110].
The LNP-mRNA cargos reach muscle cells by endocytosis shortly after injection, and the mRNA is subsequently translated to generate the metastable trimeric prefusion S protein.A network of blood arteries next to the muscles may later attract APCs that have already infiltrated.The use of cellular translational machinery and other cytosolic components by mRNA vaccines to produce a well-folded and completely functioning protein from each injected mRNA is one of its many advantages.A signal peptide spanning amino acids 1 through 15 is included in the translated product of mRNA vaccines that use the full-length S protein, allowing the protein to be transferred to plasma membranes or released from the cytoplasm.Class I major histocompatibility complexes (MHCs) will include the bulk of the protein once it has been mostly broken down by endosome-derived proteasomes and presented to CD8 + and CD4 + T cells, respectively [111].The class II MHC complex is assembled by DCs transfected with an mRNA vaccine or its endocytosed immunogens and then presented to immune cells.However, humoral immune response via B cell activation is the primary method of vaccination with an mRNA vaccine.Naïve B cells will multiply and develop into memory B cells or AB-secreting plasma cells in lymphoid organs after being activated by ligation of CD40 and interaction with cognate CD4 + T cells.Affinity determines whether a freshly activated B cell will develop into a long-lived plasma cell or a dormant memory B cell.Plasma cell-produced ABs circulate in the blood and bind and neutralize antigens upon secondary antigen exposure, preventing the antigen-carrying virus from infecting its target cells.Without enough ABs, memory B cells won't be triggered to create a subsequent immune response [112].The mRNA vaccines also have a self-adjuvanting property.Myeloid differentiation marker 88 (MyD88) signaling is activated when ssRNA is identified by TLR7 and TLR8 in endosomes.TLR3, retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and other molecules recognize dsRNA and trigger the activation of TIR-domain-containing adapter-inducing interferon-β (TRIF) and mitochondrial antiviral signaling protein (MAVS) molecules, which in turn mediate the production of type-I interferons IFN AB formation, specific cellular immune responses, and selfadjuvant effects are often induced by mRNA vaccines through the processes as mentioned above [113][114][115] (Fig. 3).

mRNA vaccine carriers with their advantages and challenges
Getting messenger RNA vaccines into human cells efficiently is a huge challenge.After entering the body, naked mRNA is quickly broken down by nucleases because it is an external nucleic acid.The immune system has little trouble recognizing it.Naked mRNA as a vaccine has far fewer pharmacological effects.Protecting given mRNA from nucleases and facilitating transport into cells are necessary for mRNA vaccines to enhance immunological effectiveness [117].Recent developments have elevated the ambitions of mRNA as a vaccine platform.As an illustration, protein synthesis in vivo was significantly enhanced through chemical modifications of RNA employing nucleotide analogs, such as pseudouridine, which mitigated the translation inhibition induced by the unmodified nucleotides.The application of highperformance liquid chromatography (HPLC) purification enhanced the translation efficacy and purity of mRNA by eliminating in vitro transcription byproducts, including dsRNA, which had the potential to impede mRNA translation.Lipids and LNPs have been employed to transport small-molecule pharmaceuticals and siRNAs.The utilization of LNPs for mRNA delivery significantly improved the in vitro and in vivo delivery efficiency of mRNA.Implementing novel formulation technologies, including continuous-flow microfluidic devices, facilitated the consistent synthesis of NPs in arbitrary dimensions and scales [118,119].Four essential components comprise the LNPs in mRNA COVID-19 vaccines.Cholesterol, a neutral phospholipid, a polyethylene-glycol (PEG) lipid, and an ionizable cationic lipid contain these.Positively charged (at low pH) ionizable amine groups are present and can interact with negatively charged mRNA during particle formation.This facilitates membrane fusion throughout import.Furthermore, PEG-lipid serves the purpose of regulating particle size and acting as a steric barrier to inhibit storage-related aggregation.These elements, in conjunction with the mRNA, generate particles measuring between 60 and 100 nanometers through a rapid blending production method.For instance, the nCoVsaRNA and ARCoV vaccine candidates for SARS-CoV-2 have mean particle sizes of 75 and 89 nm, respectively.Present mRNA-LNP COVID-19 vaccines have the disadvantage of requiring storage at (ultra)low temperatures.By identifying the underlying cause of these vaccines' instability, it may be possible to enhance the stability of mRNA-LNP products, thereby facilitating the storage of vaccines at lower temperatures [120].
Initially, cationic liposomes were implemented as liposome delivery systems in mRNA vaccines.Liposomes are spherical vesicles that consist of phospholipids arranged in either a single or multiple layers.Composed of materials that typically include polar head groups and nonpolar tails, the vesicle possesses an aqueous interior that harbors the target gene.Vesicle formation is stimulated by the hydrophobic and hydrophilic interactions between these groups.Electrostatic interactions enable positively charged cationic lipids to assemble with negatively charged mRNA to produce a lipoplex (LP), a multilayer cystic complex.Encapsulated mRNA in the LP is resistant to RNase degradation, allowing for its delivery without degradation.Nevertheless, due to their positive charge in physiological environments, cationic lipids are susceptible to interactions with other negatively charged molecules in biological fluids.Furthermore, they are readily captured by immune cells, which ultimately compromises their delivery capabilities.pH-responsive cationic lipids are screened and fabricated into various mRNA delivery vehicle structures on this basis [121].
For many years, cationic polymers such as poly(Llysine), polyethyleneimine (PEI), DEAE-dextran, poly(βamino esters) (PBAE), and chitosan have been utilized extensively for the transport of nucleic acids.In its most basic form, electrostatically bound cationic polyplexes are created when excess cationic polymers are combined with nucleic acid.Despite the large number of polymers that have been made, they are not as sophisticated as LNPs in terms of delivering nucleic acids, and there are relatively few studies on animals that have effectively used them to provide vaccines.Co-forming PBAEs with polyethylene glycol (PEG)-lipids produced mRNA/PBAE/ PEG-LNPs.Through intravenous injection, these NPs showed that they could deliver mRNA to animal lungs.A biodegradable polymer called poly(amine-co-ester) (PACE) terpolymer was studied using erythropoietin as a reporter in the context of post-intravenous gene delivery [122].
As an initial delivery reagent for in vitro transcribed (IVT) mRNA, diethylaminoethyl (DEAE) dextran was examined.DEAE-dextran is one hundred to one thousand times less effective at transfecting mRNA than lipid-mediated transfection, according to subsequent research.This discovery also facilitated the development of lipid-based transfection reagents for nucleic acids, including mRNA, thereby impeding the progress  [116] of polymeric carriers.A comprehensive investigation was conducted evaluated the functional and antigen-specific T-cell responses after mRNA delivery.To achieve this, the polymers polyethylenimine (PEI) and PBAE were compared to the commercial transfection reagent Lipo-fectamine™ 2000 and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).mRNA complexed with the gag HIV-1 antigen is present in every carrier.In the spleen and lymph nodes of mice immunized with gag mRNA complexed with cationic lipids, IFN-γ-secreting T cells specific for gag were identified.However, this was not the case with mice vaccinated with bare or polymercomplexed mRNA.PEI and its derivatives are cationic polymers that are frequently utilized.These substances exhibit water solubility, a substantial positive charge density linked to the amino groups, and have been validated as mRNA carriers for in vitro transfection.PEI is hazardous because it has a high molecular weight, namely more than 25 kDa.The probable reason for this phenomenon is the attachment of anionic serum proteins to the surface of the polyplex, which occurs due to the interaction between cationic polymers and anionic serum plasma proteins.However, the subsequent increase in size is temporary as the proteins that adhere to the surface of the polyplexes ultimately prevent the aggregation of particles.Numerous endeavors have been undertaken to alleviate these difficulties.The initial demonstration of the feasibility and effectiveness of cationic polymer-mediated mRNA vaccine transfection was accomplished via intranasal administration of cyclodextrin-covalent 2 kDa PEI.The conjugation of cyclodextrin to PEI facilitated the displacement of the charge density along the backbone of the polyamine, leading to a decrease in cytotoxicity while simultaneously preserving protonatable groups, which ultimately enhanced transfection [123][124][125].

mRNA vaccines in Mpox infection
mRNA and multi-epitope-based vaccines (MVC) against Mpox were designed using proteomics and structural vaccinology methods in this study.First, researchers isolated 10 proteins from the Mpox proteome that may be used as vaccine targets.Epitopes of these proteins were then mapped using structural vaccinology methods for B cells, cytotoxic T lymphocytes (CTLs), and Helper T lymphocytes (HTLs).Epitopes from 9 CTL, 6 B cells, and 5 HTL were combined using appropriate linkers to create MVC (multi-epitope vaccine) and mRNA-based vaccines.Efficient expression in the E. coli K12 strain and a strong interaction between the proposed MVC and TLR2 were found using molecular docking, binding free energy calculation, and in silico cloning.The results of the immune simulation showed that the antigen titer after the injection peaked on day 5 and then rapidly declined upon the production of IgM, IgG, IgM + IgG, DCs, IFN-gamma, and IL (interleukins), indicating that the designed vaccine candidate may be effective at inducing an immune response against Mpox.Optimizing the vaccine architecture by changing the linkers and the adjuvant attachment may further enhance the immune response against Mpox, although this has not been tested in the present investigation.As a result of a study, highly antigenic and non-allergenic peptides were used to create vaccines that are both productive and dynamic against Mpox [126].This research used immunoinformatics methods to create Mpox vaccines models based on mRNA.To better anticipate T-and B-cell epitopes, three proteins were selected based on their antigenicity, allergenicity, and toxicity scores.To improve immune responses, vaccine constructions were designed using primary T-and B-cell epitopes connected with epitope-specific linkers and adjuvants.To create a vaccine that is both stable and highly immunogenic, the Kozak sequence, MITD sequence, tPA sequence, Goblin 5' and 3' UTRs, and a poly(A) tail were inserted.Using molecular modeling and 3D structural validation, researchers anticipated that the vaccine construct would have highquality structures.The developed vaccination model's more excellentprotection against numerous Mpox pathogenic strains was hypothesized based on population coverage and epitope-conservancy.After considering several docking scores and physicochemical and immunological factors, Mpox-V4 was the most promising.The best vaccine model was projected to have high structural stability and binding affinity with immune receptors to induce cellular and humoral immunogenic responses against Mpox by molecular dynamics and immunological simulation assessments.Further research on these top-priority constructions in the lab and humans might pave the way for a Mpox vaccine that is both effective and safe [127].
Two Mpox quadrivalent mRNA vaccines (mRNA-A-LNP and mRNA-B-LNP) were developed in a separate investigation.The Mpox-specific antigens A29L, A35R, M1R, and B6R inspired the development of these vaccines.Mice are prompted to produce IgG ABs specific to Mpox and neutralizing ABs specific to VACV after receiving a double intramuscular injection of mRNA-A-LNP and mRNA-B-LNP.These vaccines not only have a strong cellular immune response but also a lasting effector memory T and germinal center B cell reactivity in mice, with a bias against Mpox.Additional protection against VACV challenge in mice is provided by dual administration of mRNA-A-LNP and mRNA-B-LNP.Nude mice are protected from the VACV challenge when passively transported sera from mRNA-A-LNPand mRNA-B-LNP-immunized mice are administered.Researchers in an in vivo study demonstrated Mpox quadrivalent mRNA vaccine was safe safety since neither mRNA-A-LNP nor mRNA-B-LNP caused any significant adverse effects.The injection location did not show any signs of aberrant skin responses, according to the study.Because the vaccine does not cause a major cutaneous reaction (also known as "take"), there is no chance of autoinoculation or accidental vaccination, which is in contrast to ACAM2000 (the damage at the vaccine site is often used as a marker of successful vaccination in highly replicating vaccines like ACAM2000).These mRNAbased vaccine show promise as a preventative measure against not just Mpox but also other orthopoxviruses like smallpox [128].
In the US, scientists created a multivalent mRNA vaccine (Mpoxac-097) against Mpox and tested its immunogenicity in mice.The tandem 2 A peptides that connect the five Mpox viral antigens (A29L, E8L, M1R, A35R, and B6R) were then codon-optimized.This vaccine protects against a VACV challenge and produces a robust immune response, including nABs and a T-cell response specific to Mpox.Mice given Mpoxac-097 did not exhibit significant pathological changes.In conclusion, the immunogenicity of Mpoxac-097, Mix-5, and single antigen LNP mRNAs were compared to those of multivalent Mpox mRNA vaccine candidates.The VACV challenge may be avoided after receiving an Mpoxac-097 vaccine since it produces broadly nABs and an Mpox-specific T-cell response.Mpoxac-097 is as effective and as immunogenic as Mix-5.Antigen tandem co-expression is still enticing because of its less complicated manufacturing process.The immunization of mice with Mpoxac-097 did not result in any significant pathologic alterations.Taken together, researchers' results suggest that a multivalent Mpox mRNA vaccine is feasible [129].A35R-M1R fusions (VGPox1 and VGPox2) and a combination of encapsulated full-length mRNAs for A35R and M1R (VGPox3) were generated as part of a separate investigation expressing Mpox proteins M1R and A35R.Anti-A35R total IgGs were detected as early as day 7 after a single immunization with all three vaccines.Anti-M1R total IgGs were created quickly after vaccination with VGPox 1 and 2; however, it took until day 35 for VGPox 3 to exhibit any substantial anti-M1R ABs.nAB and the T cell immunological response showed similar patterns.Mice exposed to a fatal dose of the virus were protected against infection, and the virus was eliminated from their lungs after vaccination with either of the mRNA vaccine groups.These data suggest that compared to coexpression of the two separate proteins, the new mRNA vaccines encoding a fusion protein of A35R and M1R elicited stronger anti-virus immunity.For protection against the Mpox virus, mRNA vaccines are as effective as the present whole-virus vaccines, if not more so [130].The development and synthesis of a panel of multicomponent Mpox vaccine candidates, which express various combinations of viral antigens such as M1R, E8L, A29L, A35R, and B6R, were conducted by researchers.This was achieved by the use of the LNP-encapsulated mRNA vaccine platform, which was pioneered by Zhang et al.The administration of two doses of mRNA vaccine candidates in mice has induced a robust AB response and a particular T-helper 1 (Th1)-biased cellular response.This has been demonstrated by in vitro and in vivo characterization.The mice that received vaccinations with the penta-and tetra-component vaccine candidates, namely AR-Mpox5 and AR-Mpox4a, exhibited enhanced protection against the VACV challenge.The mRNA vaccine candidates of Mpox showed efficacy in mitigating weight loss in BALB/c mice after a high dose of VACV infection.Before conducting clinical trials, it would be advantageous to do more research to evaluate the efficacy of the preventive measures in other animal models, such as the non-human primate challenge model using the circulating Mpox strain.Significant antigen-specific CD8 + T cell responses were seen in the AR-Mpox5 and AR-Mpox4b vaccinated groups after administration of the multicomponent mRNA vaccines.The multicomponent mRNA vaccines induced an immunological response in CD4 + T cells that was inclined towards generatingTh1 cytokines when exposed to various antigens.Similarly, cynomolgus macaques exhibited a Th1-biased antigen-specific cellular immune response after receiving three doses of a multivalent smallpox DNA vaccine.Conversely, mice inoculated with MVA had a substantial augmentation in the population of CD8 + T cells that produced IFN-γ [131].

Orthopoxviruses and Mpox mRNA-based vaccine
The study demonstrated that the vaccine formulated in this research, targeting Mpox, VARV, and VACV, effectively induced robust humoral and cellular immune responses with repeated vaccinations using in silico vaccine techniques.Following in silico vaccine with the proposed vaccine, the B cell population underwent activation, resulting in enhanced production of immunoglobulins, CD8 T-cytotoxic and CD4 Th cells, memory cells, and cytokines.The vaccine was shown to be stable and exhibit ideal qualities based on physicochemical criteria, and molecular docking revealed good binding to MHC molecules.Due to the genetic similarities of antigens in this work, an mRNA vaccine targeting conserved epitopes common to all three viruses was developed.The selection of antigens A29, A30, A35, B6, and M1 was made to develop a universally applicable mRNA-based immunization.The multi-epitope mRNA construct was developed using B and T cell epitopes found in the conserved areas.These epitopes were determined by comparing the sequences of Mpox, VACV, and VARV.The vaccine construct was shown to be stable and to bind to MHC molecules optimally by immunoinformatic analysis.Immune simulation analyses triggered humoral and cellular immune responses.Based on in silico research findings, it is suggested that the universal mRNA multiepitope vaccine candidate developed in this study can potentially protect Mpox, VARV, and VACV.This might have significant implications for the advancement of strategies aimed at mitigating the occurrence of potentially devastating pandemics [132].Extensive research on VACV has shown that some of its antigens, including A27, L1, A33, and B5, are very comparable to the homologous antigens of other orthopoxviruses regarding immunogenicity.These results established the feasibility of using VACV antigens as vaccine targets to create a universal poxvirus vaccine.In the realm of scientific investigation, a group of scholars employs a quartet of distinct vaccinia viral antigens, including A27, L1, A33, and B5, to fabricate a novel vaccination candidate for poxvirus, denoted as mRNA-ALAB-LNP.Following administration of a singular vaccine, mice exhibited robust production of anti-L1 ABs while displaying comparatively diminished immune responses against A33, A27, and B5.All IgG titers were over 5 logs after the second injection, with anti-A33 IgG having the highest titer of the four antigens.The high binding AB level demonstrated a strong neutralizing ability against the VACV.Among the four tested antigens, only A33 was demonstrated to cause a significant cellular response to IFN-γ.strong levels of cross-reactivity were shown by the fact that serum IgG responses to matching Mpox antigens A35, M1, A29, and B6 were as strong as, or even higher than, responses to vaccinia antigens.This suggests that the mRNA-ALAB, which encodes four vaccinia antigens, might be a promising candidate for future vaccine development against infection with Mpox, smallpox, and other orthopoxviruses [133].The other aimed to create a multivalent mRNA vaccine for Mpox that would target the EV and MV surface proteins and analyze its effectiveness and molecular basis for protection.The immunogenicity of four mRNA vaccines containing various combinations of EV (A35R and B6R), MV (A29L, E8L, H3L, and M1R), or EV and MV surface proteins was evaluated in BALB/c mice.Seven days after the first vaccine, a dynamic immune response was seen, and after two vaccinations, a robust IgG response to all immunogens was confirmed by ELISA.The observed correlation between a higher cumulative IgG response and the associated neutralizing activity against VACV, resulting from exposure to a larger number of immunogens, highlights the cumulative effect of each immunogen in stimulating an immune response and eradicating VACV infection.In addition, the mRNAbased vaccines elicited a CD4 + T cell response skewed towards the Th1 phenotype and specific to the antigen.The efficacy of mRNA vaccines containing different combinations of EV and MV surface antigens in protecting a mouse model against a lethal VACV challenge was investigated.Results indicated that the vaccine formulation comprising EV and MV antigens demonstrated the highest level of protection.The findings of this study provide insights into the protective mechanism of MPV multi-valent mRNA vaccine and provide a foundation for developing enhanced and safer mRNA vaccines to mitigate future outbreaks of Mpox [134].According to the authors of the research, the mRNA-LNP vaccine demonstrated the ability to elicit immunity specific to Mpox and provide cross-protection against a deadly VACV challenge.The vaccine contains a combination of four Mpox surface proteins that are known to be highly conserved and play crucial roles in the processes of viral attachment, entry, and transmission.The administration of mRNAbased vaccines resulted in a more pronounced Fc-effector Th1-biased humoral immune response towards the four antigens of Mpox and the four homologs of VACV.Additionally, these vaccines exhibited enhanced neutralizing and cellular spread-inhibitory properties against both Mpox and VACV.This finding contrasts the existing Mpox vaccine, which utilizes the MVA platform.The administration of mRNA vaccines expressing two, three, or four Mpox antigens has shown efficacy in preventing disease-associated weight loss and death.However, single Mpox antigen mRNA vaccines only provided partial protection against the VACV challenge.Multivalent Mpox mRNAs, which are linked with both neutralizing and non-nABs activities, provided remarkable crossprotection that outperformed homologous protection by MVA.These results show that an mRNA-based vaccine targeting four highly conserved viral surface antigens may protect against VACV by inducing highly functional ABs that can swiftly reduce viral infection [135].Similar to what was done by Xia et al. (Homologs to VACV A27, A33, B5, and L1), a panel of mRNA-LNP-based vaccine candidates encoding a set of four highly conserved Mpox surface proteins implicated in viral attachment, entry, and transmission has been developed by Xia et al.Administration of these antigenic mRNA-LNPs either separately (5 µg each) or in an average combination at a low dosage (0.5 µg each) evoked Mpox-specific IgG ABs and strong VACV-specific nABs when given twice, despite possible variations in immunogenicity among the four antigenic mRNA-LNPs.Infection with VACV led to weight loss and death in mice, whereas mice given two 5 µg doses of A27, B5, and L1 mRNA-LNPs, or an average mixture of the four antigenic mRNA-LNPs (each dosage was 5 µg), were protected.The data they gathered support the idea that these antigenic mRNA-LNP vaccine candidates might be useful in the fight against Mpox and similar orthopoxvirus infections [136].Sang et al. developed two Mpox quadrivalent mRNA vaccines, namely mRNA-A-LNP and mRNA-B-LNP, in a distinct investigation.These vaccines were manufactured using two IMVs (A29L and M1R) and two EEVs (A35R and B6R).Following administering two intramuscular injections of mRNA-A-LNP and mRNA-B-LNP, mice demonstrated the ability to generate Mpox-specific IgG ABs and robust VACV-specific nABs.In mice, the immune response to Mpox included the induction of memory B-cell immunity alongside killer memory T-cell immunity.The protection of nude mice against the VACV challenge was achieved by the passive transfer of sera obtained from vaccinated mice exposed to mRNA-A-LNP or mRNA-B-LNP.Mice that received both mRNA-A-LNP and mRNA-B-LNP at different doses exhibited comparable resistance to the VACV challenge.The safety and efficacy of mRNA-A-LNP and mRNA-B-LNP as potential vaccines against Mpox epidemics and other outbreaks produced by orthopoxviruses, including the smallpox virus, were shown by researchers [137].
Researchers created two multi-antigen mRNA vaccine candidates, which encode four (M1, A29, B6, A35, referred to as Rmix4) or six (M1, H3, A29, E8, B6, A35, referred to as Rmix6) Mpox antigens, by using a streamlined manufacturing technique of combining DNA plasmids before transcription.Researchers found that whereas Rmix6 produced noticeably greater cellular immune responses than Rmix4, those Mpox multiantigen mRNA vaccine candidates had similarly effective cross-neutralizing immunological responses against VACV.Encasing each mRNA that codes for an antigen individually is a common approach for developing multiantigen mRNA vaccines, even though covering all the mRNAs at once is more efficient.It is straightforward to confirm the immunogenicity of each mRNA vaccine component thanks to the distinct LNP production.Furthermore, mice that received vaccinations with both vaccine candidates were shielded from the deadly VACV challenge.The M1 antigen effectively induced nAB responses, according to an analysis of the B-cell receptor (BCR) repertoire elicited by the Mpox individual antigen.Furthermore, all nABs among the top 20 frequently occurring ABs seemed to target the same conformational epitope as 7D11, suggesting a potential vulnerability to viral immune evasion.According to these results, Rmix4 and Rmix6, which come from a streamlined production method, seem like good options to fight Mpox [138].(Table 2).

Future perspective
The results of a clinical modeling investigation show little protective immunity against Mpox.This indicates that at the present stage of the Mpox re-emergence, most individuals are in danger of contracting the virus because they lack immunity to the disease.Mpox recurrent infectivity might provide a challenge [139].Worldwide, there have been over 80,000 verified instances of Mpox, and those who have recovered are thought to be immune to reinfection.Nonetheless, there has been a recent case of a person who seems to have reinfected.Researchers reported two cases of possible Mpox reinfection at San Raffaele Hospital in Milan, Italy, in this Comment.These instances indicate two possible reinfections with the Mpox.Researchers detected high cycle threshold values, transient symptoms, clinical features like those of Mpox, and detectable nABs for both of the second episodes after the virological and clinical healing of the initial episodes.For Mpox with a recent start, the cycle threshold values were high, indicating low viral levels.Other theories that might account for reinfection include sexual contamination or relapse from tissue reservoirs.Co-infections in other individuals may have contributed to or worsened symptoms, or they may have made reinfection easier.Also, Investigators isolated and sequenced Mpox from both patients from samples collected during the first episodes.They could not isolate the virus from samples from the second episode, probably due to low viral loads as indicated by the high cycle threshold values.SARS-CoV-2 could also have had a negative influence.Although genomic data cannot confirm the presence of two distinct viruses, and thus reinfection (in contrast to relapse, which would have presented with the same virus), clinicians need to be aware of potential Mpox reinfections and should investigate with viral culture and sequencing.Furthermore, the potential of Mpox reinfection has implications for transmission and vaccination policies [140].In addition, the situation might become much more dire if specific mutations occur in Mpox, reducing the efficacy of the smallpox vaccine against Mpox.Although the number of confirmed cases of Mpox each day is falling, the virus is still present worldwide and might return with the same clade or spread to other regions, including Central Africa.The field of vaccinology has been significantly transformed by the advent of mRNA-based vaccines, owing to their exceptional safety profile, cost-effectiveness in production, high potency, and rapid development [103,132,141].Clinical trials have provided evidence to support the safety and efficacy of the two predominant mRNA vaccines against SARS-CoV-2 and its many variations [101].Presently in use, JYNNEOS is a live vaccine derived from the attenuated, non-replicating orthopoxvirus strain-modified vaccinia Ankara-Bavarian Nordic (MVA-BN).Unlike other vaccines, MVA-BN is incapable of replicating within the human body.Moreover, it elicits humoral and cellular immune responses specific to orthopoxviruses without severe adverse effects.Based on the observations above, the effectiveness of currently available vaccines remains dubious.It is widely believed that it fails to deliver the The first and second dosages are given four weeks apart, using the same default simulation settings.
The antigen titer after the injection peaked on day 5, and then rapidly declined upon the production of IgM, IgG, IgM + IgG, DCs, IFN-gamma, and IL (interleukins), indicating that the designed vaccine candidate may be effective at inducing an immune response against Mpox. [126] T-and B-cell epitopes connected with epitope-specific linkers and adjuvants.To create a vaccine that is both stable and highly immunogenic, the Kozak sequence, MITD sequence, tPA sequence, Goblin 5' and 3' UTRs, and a poly(A) tail were inserted.Two doses of 5 µg of A27, B5, and L1 mRNA-LNPs or a 2 µg average mixture of the four antigenic mRNA-LNPs protected mice against weight loss and death after the VACV challenge.The VACV challenge may be avoided after receiving an Mpoxac-097 vaccine since it produces broadly nABs and an Mpox-specific T-cell response.Mpoxac-097 is as effective and as immunogenic as Mix-5.Antigen tandem co-expression is still enticing because of its less complicated manufacturing process.
[ These data suggest that compared to co-expression of the two separate proteins, the new mRNA vaccines encoding a fusion protein of A35R and M1R elicited stronger anti-virus immunity.For protection against the Mpox virus, mRNA vaccines are as effective as the present whole-virus vaccines, if not more so.[130] Multicomponent Mpox vaccine, which expresses various combinations of viral antigens such as M1R, E8L, A29L, A35R, and B6R.

AR-Mpox5 and AR-Mpox4a
In vivo (C57BL/6mice) Intramuscular administration of 5 µg of each antigenencoded mRNA was performed, followed by a three-week augmentation with the identical dose.
The mRNA vaccine candidates of Mpox shown efficacy in mitigating weight loss in BALB/c mice after a high dose of VACV infection.Significant antigen-specific CD8 + T cell responses were seen in the AR-Mpox5 and AR-Mpox4b vaccinated groups after administration of the multicomponent mRNA vaccines.The multicomponent mRNA vaccines induced an immunological response in CD4 + T cells that was inclined towards generating Th1 cytokines when exposed to various antigens. [131] Table 2 Efficacy and performance of several mRNA-based vaccines in preventing Mpox infection anticipated and necessary effectiveness in containing the disease's transmission, in addition to the recurrent incidence of severe complications associated with the vaccine that may exceed the morbidity and mortality caused by Mpox.Consequently, there is an urgent requirement to develop novel vaccines that exhibit enhanced specificity, safety, and efficacy against Mpox.Regarding this, researchers advise the development and testing of more recent slain and/or mRNA vaccines to surmount the drawbacks of the existing ones before the declaration of Mpox as a pandemic and the Implementation of preparedness activities becomes more difficult.However, in the interim, until such vaccines undergo testing and become commercially available, the MVA-BN vaccine can be utilized to mitigate the transmission of the disease due to its superior efficacy and safer profile in comparison to the ACAM2000 vaccine [65].The population's resistance against SARS-CoV-2 is declining due to new variations being selected, which has led to the redesign of mRNA vaccines.The benefit of mRNA vaccines is that they may be quickly modified by altering the immunogenic transgene to target variations.Conventional vaccines usually involve the cultivation of substantial quantities of active viruses followed by their inactivation, which may span many weeks or months.On the other hand, mRNA vaccines can be mass-produced, tested, and created fast [142].Administration of these antigenic mRNA-LNPs either separately (5 g each) or in an average combination at a low dosage (0.5 g each) evoked Mpox-specific IgG ABs and strong VACV-specific nABs when given twice, despite possible variations in immunogenicity among the four antigenic mRNA-LNPs. [136] These vaccines were manufactured using two IMVs (A29L and M1R) and two EEVs (A35R and B6R).Mice demonstrated the ability to generate Mpox-specific IgG antibodies and robust VACV-specific neutralizing antibodies.In mice, the immune response to Mpox included the induction of memory B-cell immunity alongside killer memory T-cell immunity.The protection of nude mice against the VACV challenge was achieved by the passive transfer of sera obtained from vaccinated mice exposed to mRNA-A-LNP or mRNA-B-LNP. [137]

Table 2 (continued)
experimental APSV, a potential alternative intervention [143].In the US, there are now two vaccinations that have been developed to mitigate the risk and severity of Mpox infection.These vaccines are known as JYNNEOS (sometimes referred to as Imvamune or Imvanex) and ACAM2000.JYNNEOS has emerged as the vaccine of choice for the ongoing Mpox epidemic [144].Although encapsulating all mRNAs simultaneously is preferable for multi-antigen mRNA vaccine production, isolating and encapsulating each mRNA encoding an antigen is the standard practice.Using the individual LNP preparation, the immunogenicity of each component of an mRNA vaccine may be quickly and easily tested.Researchers further demonstrated that the Mpox multi-antigen mRNA vaccine candidates could induce robust cross-neutralizing immune responses against VACV.Furthermore, it was shown that Rmix6 induced significantly higher cellular immune responses compared to Rmix4 [138].mRNAbased vaccines provide several potential advantages over standard vaccines, such as the following: In addition to eliciting humoral and cell-mediated immune responses, these vaccines possess several advantageous characteristics.Firstly, they lack infectious components and pose no risk of stable integration into the genome of host cells.Secondly, they are well-tolerated by individuals in good health.Thirdly, they are cost-effective and can be rapidly manufactured using easily standardized and scalable procedures.Fourthly, they enhance the ability to respond effectively to widespread emerging outbreaks.Lastly, they exhibit a high level of tolerability [103].For instance, vaccines are of significant importance in preventing contagious illnesses, and the application of mRNA vaccine technology in safeguarding against COVID-19 demonstrated its efficacy and safety as a platform.Using the Mpox mRNA sequences A29L, M1R, A35R, and B6R as inspiration, scientists in an investigation created two Mpox mRNA vaccine candidates known as MPXfus and MPXmix.The MPXfus was a unicomponent system consisting of four antigen proteins that were fusion-linked in tandem via a flexible linker and encoded as a fusion protein by a single mRNA.The MPXmix was a multicomponent comprising four mRNA molecules, each corresponding to a specific antigen protein.Lignum NPdelivered MPXfus or MPXmix of equivalent quality was used to immunize mice to assess and contrast the immune responses elicited by these two potential Mpx vaccines.Both MPXfus and MPXmix could elicit a robust cellular immune response and a high level of antigenspecific ABs in mice, according to the results of immune response analyses.Furthermore, the outcomes of virus neutralization assays indicated that sera obtained from rodents immunized with MPXfus or MPXmix exhibited significant neutralizing activities in the presence of the VACV.Furthermore, there was no significant difference observed in the titers of antigen-specific ABs, levels of cellular immune response, or activities of nABs against the VACV induced by MPXfus and MPXmix [145].Three mRNA vaccines are created in different research that encode the Mpox proteins A35R and M1R.These vaccines include fusions of the A35R extracellular domain with the M1R protein (VGPox 1 and VGPox 2) and a combination of encapsulated full-length mRNAs for both A35R and M1R (VGPox 3).This work showed that in terms of anti-viral immunity, mRNA vaccines producing fusion proteins made of Mpox A35R extracellular domain with a signal peptide and M1R are superior to the sublethal live VACV-WR virus.The population was protected against the deadly VACV challenge due to the early induction of humoral immunity against the virus by VGPox 1 and VGPox 2, which occurred as early as 7 days post-vaccination.The strong similarity between Mpox and vaccinia in researchers' results makes them more remarkable.This suggests that VGPox 1 or 2 might be effective mRNA vaccines against additional orthopoxviruses.Although this work offers insightful information on the effectiveness of mRNA vaccines against Mpox, many limitations should be noted.For example, researchers conducted in vitro neutralization and in vivo tests using VACV, not Mpox.Further research comparing the mRNA vaccine to other licensed vaccines in non-human primates, such as JYNNEOS, might build on these results [146].It is crucial to keep evaluating the safety and efficacy of the current vaccines to prevent Mpox.As a result, creating a new vaccine with broad application potential for Mpox and its growing variations may provide a useful preventative strategy to address the infection's dynamic nature.Numerous studies have shown that mRNA-based LNP is superior to conventional vaccination techniques because it produces higher levels of nABs, more potent Mpox-specific T-cell responses, and protection against Mpox.The safety profiles of lipophilic medications are dependent on dosage and combination, and it is not possible to predict long-term adverse effects, especially after many doses, since long-term health outcomes data are not currently available.Advancements in research should be conducted to reevaluate the benefits vs. risks before mRNA vaccines are widely used in low-risk patients who may need life-threatening treatment.In addition, it is important to comprehensively assess several facets of vaccination effectiveness, including reactogenicity, cytotoxicity test outcomes, safety, and adverse reactions, with a specific focus on those who are at heightened risk or are more vulnerable.This evaluation is essential for discerning the optimal choice between conventional and novel vaccines in terms of efficacy [144].The surface antigens encoded by the BNT166 vaccine candidates are present in both infectious forms of Mpox, enabling them to combat virus replication and infectivity effectively.The safety, tolerability, reactogenicity, and immunogenicity of two mRNA-based multivalent vaccine candidates for active immunization against Mpox will be assessed in the clinical trial (NCT05988203).Substudy A is a Phase I, openlabel, dose-escalation trial that aims to evaluate the safety, immunogenicity, and reactogenicity of two multivalent vaccine candidates (BNT166a and BNT166c) at up to three dose levels in approximately 64 healthy participants who have not been vaccinia-naïve or have no prior history of smallpox vaccination.Two doses will be administered approximately 31 days apart.If the sponsor chooses not to activate the group with BNT166c, randomization will not occur.Substudy B, which is a randomized, observer-blinded, and sponsor-unblinded Phase I substudy, aims to evaluate the safety, immunogenicity, and reactogenicity of two multivalent vaccine candidates (BNT166a and BNT166c) in approximately 32 healthy participants who have previously been vacciniaexperienced with smallpox.Two doses will be administered approximately 31 days apart.The participants will be assigned in a 1:1 randomization.If the sponsor chooses not to activate one of the groups, this substudy will be an open-label substudy with a single group.The objective of the Phase 1/2 trial is to recruit 196 healthy participants who are either vaccinia-naive or have no prior history of known or suspected smallpox vaccination [89,147].The development of mRNA vaccines is a swiftly evolving field.Numerous mRNA vaccines are undergoing clinical trials for various diseases, including cancer and influenza.A critical obstacle associated with mRNA vaccines is their inability to withstand extreme cold and thermostability.The fact that a thermostable vaccine (CvnCoV) developed by CureVac exhibited a clinical efficacy of less than 50% suggests that the current objective is to create a thermostable vaccine that demonstrates "high clinical efficacy." In light of the lack of stability data on mRNA vaccines in the scientific literature, it is necessary to conduct well-designed mechanistic studies utilizing extended periods, various excipients, and storage temperatures to fill the existing knowledge gaps.Based on the scant information at investigators' disposal, they have deduced that the vaccines' lack of thermostability can be attributed to the difficult characteristics of the synthetic mRNA employed in them [148].Additionally, the thermostability of mRNA vaccines can be enhanced, with or without substantial formulation modifications, according to the researchers' findings.Alternative techniques for dehydrating mRNA LPN suspension, such as lyophilization, will serve as feasible alternatives to enhance the stability and storage conditions of mRNA vaccines.The second way to improve the thermostability for vaccines is through mRNA sequence optimization.Following the initial lack of success in their first thermostable vaccine clinical trial, CureVac is presently engaged in the development of a second COVID-19 vaccine that incorporates noncoding region mRNA optimization.Furthermore, including excipients, such as lipids and cholesterol in LPN, may render the vaccine susceptible to oxidative degradation.Consequently, by optimizing the NP components and manufacturing process, it is possible to enhance the stability of vaccines.Lastly, a theoretical study proposes that repurposing mRNA to generate double-stranded regions may represent an additional strategy for improving vaccine stability [149].Several low-income and middle-income nations face significant unmet demand for mRNA vaccines due to inadequate accessibility.This indicates that accessibility remains a substantial determinant in low vaccination rates, surpassing the concern of vaccine reluctance.According to a study's results, global COVID-19 vaccine equity continues to be a significant concern.Concentrated endeavors to enhance vaccination rates are imperative, especially in nations with inadequate coverage and substantial unfulfilled demand for immunizations.It is important to mitigate obstacles to vaccine access, guarantee the availability and dissemination of mRNA vaccines, and surmount vaccine hesitancy to decrease unfulfilled demand and attain greater vaccination rates in diverse geographical areas [150].

Conclusion
The significance of researching the development of targeted vaccine candidates and the exploration of therapies for Mpox should not be underestimated, given that the virus exhibits lower infectivity compared to SARS-CoV-2, and the smallpox vaccine has shown efficacy against Mpox.Current pandemics, such as COVID-19, that continue to threaten global health, have brought into focus the need for affordable vaccines that can be used to protect the whole global population from infectious diseases.Furthermore, most Mpox-infected people had never been vaccinated against smallpox.The results might help governments design or advocate for public health policies and immunization programs targeting the most at risk.ABs produced responding to a smallpox vaccine are cross-reactive, meaning they can identify and fight off other orthopoxviruses.In addition, the COVID-19 pandemic highlights the need for novel biotechnology studies on the Mpox virus to spur the creation of lowcost treatments and vaccines.Last but not least, given the potential for severe illness and death caused by the present epidemic, it is critical that rigorous, well-controlled, prospective trials be conducted immediately to demonstrate effectiveness.When dealing with the unexpected, nucleic acid platforms shine.Although success will be measured over time, the speed with which mRNA vaccines have been developed and tested since receiving FDA approval is remarkable.Nucleic acid technology might then evaluate and patent therapeutic and preventative effector molecules, like ABs.SARS-CoV-2 shows how difficult it may be to keep one step ahead of a fastadapting agent without a flexible infrastructure.mRNA vaccines may be rapidly developed, evaluated, and manufactured in large quantities.The lack of live viruses in mRNA vaccines makes them a more secure option.However, mRNA vaccines have their own set of problems.Although the vaccine's mRNA may be rapidly destroyed after injection or generate cytokine storms, allergic reactions, renal failure, heart failure, and infarction are still possible adverse effects.The delivery and stability of such a therapy may be only beyond the horizon, given the rapid development of relevant technologies.The quick development of mRNA vaccine technologies that can have an effect during the Mpox infection is made possible by breakthroughs in bio/nanotechnology, sophisticated nano/manufacturing, and open reporting and data sharing.There is yet time before Mpox is labeled a pandemic to create a compelling and safe new generation of vaccines that eradicate the virus or develop innovative vaccine platforms.

Fig. 2
Fig. 2 The potential immunopathogenesis of Mpox infection is depicted in this diagram, including humoral immunity, innate immunity, adaptive immunity, and immune evasion.(a) The expression of chemokines (CCR5, CXCR3, and CCR6) is inhibited by the Mpox virus, which depletes NK cell function and reduces IFN-γ and TNF-α secretion.(b) Abnormalities of granulocytes and monocytes, such as basophil, eosinophil, neutrophil, and monocyte, are induced by the Mpox virus.b) Mpox may stop natural killer cells from releasing inflammatory cytokines and eliminating virus-infected cells.Mpox may also impede T cell receptor trans-signaling, disrupting the adaptive immune response.(c) By inhibiting the antiviral type 1 IFN responses, Mpox avoids the innate immune response.By decreasing mRNA production and inhibiting type-I interferon, which mediates protein kinase R phosphorylation, Mpox may evade the protein kinase R pathway.By interfering with the phosphorylation of the MAPK/ERK1/2 pathways, Mpox may also lower the generation of inflammatory mediators.By making the ATK pathway more phosphorylated, it inhibits cell apoptosis.e) Patients with Mpox are likelier to have a worse prognosis because a monoclonal AB that neutralizes the virus may enhance viral entrance into cells via the Fc region of the AB attached to the Fc receptor (FcR) on cells.A substantial T helper 2 (Th2) immune response is linked to the cytokine storm that Mpox infection causes.This response is marked by increased serum levels of IL-4, IL-6, IL-5, IL-8, and IL-10, and a decrease in Th1-associated cytokines such (IFN-α, IFN-γ, TNF-α, IL-2, and IL-12).A greater total AB titer is seen, along with increased levels of IgG and IgM[55,56]

Fig. 3
Fig. 3 NPs are based on lipids and the structure of messenger RNA.An mRNA molecule has the following components: a 5′ cap, 3′ UTR, open reading frame, poly (A) tail, and 5′ and 3′ untranslated regions (UTRs).The majority of mRNA vaccines are delivered via lipid nanoparticles (LNPs).LNPs often include lipid components such as cholesterol, phospholipids, ionizable lipids, and PEG-conjugated lipids.LNPs carrying antigen mRNA are employed to create Mpox mRNA vaccines, and the production and localization of these antigens in transfected cells are shown in Figure C. DCs and other APCs take up mRNA-LNPs or locally generated antigens.To stimulate CD4 and CD8 T cells, these APCs must first go to the lymph nodes.CD8 T cell priming leads to the production of cytotoxic T lymphocytes, which may eliminate pathogens by destroying them from the inside.T follicular helper (Tfh) cells and Th1 cells are two possible outcomes of antigen priming of CD4 T cells.Activation of a germinal center (GC) response is aided by Tfh cells.Vaccination causes GC reactions, which lead to the development of AB-secreting LLPCs and affinity-matured memory B cells (MBCs).Class flipping of antibodies (ABs) generated by LLPCs to either Th1-or Th2-associated Abs is influenced by the Tfh cell skew toward the Th1 or Th2 phenotype[116]

Table 1
Different Mpox vaccinesIn the Strategic National Stockpile, another replication-competent vaccination against the vaccinia virus (VACV) is called APSV.It is anticipated that the safety profiles of APSV and ACAM2000 will be similar.